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Expi293™ Expression SystemUSER GUIDE

For scalable transfection of Expi293F™ cells in a chemically defined, serum-freemedium, using ExpiFectamine™ 293 Transfection Kit

Catalog Number A14635

Publication Number MAN0019402

Revision B.0

Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, CA 92008For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BELIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH ORARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0019402

Revision Date Description

B.0 02 September 2020 Technical changes in recommended pH settings for growth.

A.0 29 May 2020 New user guide.

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product,you accept the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

©2020 Thermo Fisher Scientific Inc. All rights reserved.

https://www.thermofisher.com/symbols-definition

Contents

■ Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Components of the Expi293™ Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Expi293™ Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Expi293F™ Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Expi293™ Expression Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6ExpiFectamine™ 293 Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7ExpiFectamine™ 293 Transfection Enhancer 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7ExpiFectamine™ 293 Transfection Enhancer 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Opti-MEM™ I Reduced Serum Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Antibody-Expressing Positive Control Vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■ Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Procedural guidelines for Expi293F™ cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9General cell handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Guidelines for media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Thaw and establish Expi293F™ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Guidelines to thaw and establish Expi293F™ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Thaw Expi293F™ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Subculture Expi293F™ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Passage Expi293F™ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Cryopreserve Expi293F™ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Transfect Expi293F™ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Guidelines for transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Scale up transfections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Transfect Expi293F™ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Protein expression in 3L Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Recommended volumes for 3L Bioreactor transfection . . . . . . . . . . . . . . . . . . . . . . . . . 18Recommended Bioreactor settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Transfect Expi293F™ Cells in a 3L Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Expi293™ Expression System User Guide 3

■ APPENDIX A Additional guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Guidelines to optimize protein expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Plasmid DNA complexation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Harvest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Cell culture supernatant clarification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

■ APPENDIX B Positive control for transfection and expression . . . . . . . . . . . . . . . . 24

Antibody-Expressing Positive Control Vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Transfection and expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

■ APPENDIX C Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Additional products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Shaker flasks for suspension culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Orbital shaker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

CO2 controlled incubator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Plasmid purification products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Visualization and quantitation or control antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

■ APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

■ APPENDIX E Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Contents

4 Expi293™ Expression System User Guide

Product information

IMPORTANT! Before using this product, read and understand the information in the“Safety” appendix in this document.

Product description

The Gibco™ Expi293™ Expression System is a high-yield transient expression systembased on suspension-adapted Human Embryonic Kidney (HEK) cells. The Expi293™

Expression System Kit provides cells, culture medium, and reagents to transfect atotal of 1 liter production volume.

Contents and storage

For a detailed description of each component of the Expi293™ Expression System,see “Components of the Expi293™ Expression System” on page 6. For additionalordering information, see Appendix C, “Ordering information”.

Table 1 Expi293™ Expression System (Cat. No. A14635, A14635CN)

Contents Amount Storage

Expi293F™ Cells (1 × 107 cells/mL) 2 × 1 mL Liquid nitrogen[1]

Expi293™ Expression Medium 1 L 2°C to 8°C. Protect from light.

ExpiFectamine™ 293 Transfection Kit:

• ExpiFectamine™ 293 Reagent

• ExpiFectamine™ 293 Transfection Enhancer 1

• ExpiFectamine™ 293 Transfection Enhancer 2

1 kit

—

2°C to 8°C

2°C to 8°C. Protect from light.

2°C to 8°C

Antibody-Expressing Positive Control Vector (at 1 mg/mLin TE Buffer, pH 8.0)[2] 150 µg −20°C

Opti-MEM™ I Reduced Serum Medium[3] 100 mL 2°C to 8°C. Protect from light.

[1] Store the frozen cells in liquid nitrogen until ready to use. Do not store the cells at −80°C.[2] TE buffer, pH 8.0: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. [3] Opti-MEM™ I Reduced Serum Medium is a serum-free reagent.


Components of the Expi293™ Expression System

Expi293™ Expression System

The Expi293™ Expression System is a major advance in transient expressiontechnology for rapid and high-yield protein production from mammalian cells. Itis based on high density suspension culture of Expi293F™ Cells in Expi293™

Expression Medium. Transient expression is powered by the cationic lipid-basedExpiFectamine™ 293 Reagent in combination with specialized transfection enhancers.All components work in concert to generate 2- to 10-fold higher protein yieldsthan previous generation transient expression systems such as the FreeStyle™ 293Expression System. Expression yields of up to 1 gram per liter of transfectedculture have been demonstrated for some antibody and non-antibody proteins. (Visitwww.thermofisher.com/expi293 for protein expression data.)

Expi293F™ Cells

Expi293F™ Cells are human cells derived from the 293F cell line, and are a corecomponent of the Expi293™ Expression System. They are maintained in suspensionculture and optimized to grow to high density in Expi293™ Expression Medium.Expi293F™ Cells are highly transfectable and generate superior transient proteinyields compared to standard 293 cell lines.

• Growth conditions: Suspension at 37℃, 8% CO2.

• Doubling time: 24–25 hours during log phase growth. Doubling times may varybased on cell health, handling, and passage number.

• Viability: Cell viability should be greater than 90% by 7 days post-thaw.

• Subculture conditions: Grow cells to 3–5 × 106 cells/mL; then, split cells to0.3–0.5 × 106 cells/mL every 3 days or 0.2–0.4 × 106 cells/mL every four days.Do not grow above 5 × 106 cells/mL for best performance. Discard cells afterpassage number 30.

Note: We also offer fully-documented cGMP-banked Expi293F™ Cells. For orderinginformation, see Appendix C, “Ordering information”.

Expi293™ Expression Medium

Expi293™ Expression Medium is a chemically defined, serum-free, protein-free,animal origin-free medium for growth and transfection of suspension-adapted HEK293 cells. It is a core component of the Expi293™ Expression System and supportshigh density culture of Expi293F™ cell lines for scalable transient protein expression.Expi293™ Expression Medium is formulated with GlutaMAX™ Supplement, is readyto use without additional supplementation. Expi293™ Expression Medium containsno human or animal-origin components. The chemically defined formulation resultsin high lot-to-lot reproducibility and reliability. The medium is not recommended foradherent cell cultures.

Product informationComponents of the Expi293™ Expression System


http://www.thermofisher.com/expi293

Expi293™ Expression Medium exhibits the following features:

• Supports growth and transfection of Expi293F™ cells in culture formats of lessthan 1 mL in multi-well plates to greater than 10 L in disposable bioreactors

• Supports growth of suspension Expi293F™ cultures to densities over15 × 106 cells/mL.

• Transfection compatible medium enables transfection efficiencies ofapproximately 80% using ExpiFectamine™ 293 Transfection Reagent

• Enables sustained, high-level expression of high-density transiently transfectedcultures, achieving yields of up to 1 gram per liter of recombinant protein

• Does not contain phenol red

ExpiFectamine™ 293 Reagent

ExpiFectamine™ 293 Reagent is optimized for the transfection of plasmid DNA intohigh-density Expi293™ cell cultures.

ExpiFectamine™ 293 Reagent has the following features:

• Designed specifically for transfection of high-density suspension cell culture,with matching transfection enhancers that boost transfection performance andprotein expression

• Achieves protein yields 2- to 10-fold higher than other transfection reagents usedon high-density 293 cell cultures

• Employs the same transient expression protocols typically used in currentlow-density 293 suspension culture systems to easily switch from low‑densitysystems to the high yield, high-density Expi293™ Expression System

• Provides robust and reproducible transfection results

• Enables scalable transfections for culture volumes of less than 1 mL to greaterthan 10 liters, while maintaining equivalent volumetric protein yields

ExpiFectamine™ 293 Transfection Enhancer 1

ExpiFectamine™ 293 Transfection Enhancer 1 is an optimized, chemically defined,serum-free, protein-free, animal origin-free formulation designed to work inconjunction with Expi293™ Expression Medium to support, high-density transienttransfections.

Note: ExpiFectamine™ 293 Transfection Enhancer 1 may occasionally exhibit aslightly yellowish tint. However, internal studies show this has no impact on systemperformance and protein titers.

ExpiFectamine™ 293 Transfection Enhancer 2

ExpiFectamine™ 293 Transfection Enhancer 2 is a proprietary, animal origin-freeformulation developed to be used in conjunction with ExpiFectamine™ 293 Reagentand ExpiFectamine™ 293 Transfection Enhancer 1 to enhance protein production,resulting in maximal protein yields.



Opti-MEM™ I Reduced Serum Medium

Opti-MEM™ I Reduced Serum Medium is a serum-free medium used to complexplasmid DNA with ExpiFectamine™ 293 Reagent, providing superior proteinexpression through efficient transfection.

Antibody-Expressing Positive Control Vector

Antibody-Expressing Positive Control Vector is provided for transfection andexpression in Expi293F™ cells. The rabbit IgG that is produced in Expi293F™

cells after transfection with the control vector is secreted into the Expi293™

Expression Medium, with optimal yields occurring 5–7 days post-transfection. Formore information on using the Antibody-Expressing Positive Control Vector, seeAppendix B, “Positive control for transfection and expression”.



Methods

Procedural guidelines for Expi293F™ cell culture

General cell handling

• All solutions and equipment that come in contact with the cells must be sterile.Always use proper aseptic technique and work in a laminar flow hood.

• For all cell manipulations, mix the cells by gentle swirling; avoid vigorousshaking/pipetting. Cell health is critical for optimal performance.

• Expi293F™ cells are a robust cell line adapted to high-density growth conditionswith a doubling time of approximately 24 hours during log phase growth.

• For general maintenance of cells, passage Expi293F™ cells when they reach adensity of approximately 3–5 × 106 viable cells/mL (i.e., early log-phase growth),typically every 3–4 days.

Note: Cells that are subcultured at densities outside of this early log-phasegrowth window may show longer doubling times and lower titers over time.Modify the initial seeding density to attain the target cell density of 3–5 × 106

viable cells/mL at the time of subculturing.

• Use an automated cell counter or a hemocytometer with the trypan blueexclusion method to determine cell viability. Log phase cultures should be >95%viable.


Guidelines for media

Expi293™ Expression Medium is formulated with GlutaMAX™ Supplement. Forsuspension growth and transfection applications, use the Expi293™ ExpressionMedium without any supplementation.

IMPORTANT! Expi293™ Expression Medium is sensitive to light. For optimal results,use and store media protected from light.

Note: ExpiFectamine™ 293 Transfection Enhancer 1 can exhibit a slightly yellowishtint. Internal studies show this has no impact on system performance or protein titer.

Thaw and establish Expi293F™ cells

Guidelines to thaw and establish Expi293F™ cells

• IMPORTANT! On receipt, either thaw the cells immediately into pre-warmedExpi293™ Expression Medium or immediately place the frozen cells into vaporphase liquid nitrogen storage until ready to use. Do not store the cells at –80°C.

Avoid short-term, extreme temperature changes: when storing cells in liquidnitrogen following receipt on dry ice, allow the cells to remain in liquid nitrogenfor 3–4 days prior to thaw.

• Expi293F™ cell lines are supplied in a vial containing 1 mL of cells at1 × 107 viable cells/mL in 90% Expi293™ Expression Medium and 10% DMSO.

• Thaw the cells directly into Expi293™ Expression Medium, pre-warmed to 37°C.

• Before starting experiments, ensure that cells are established and frozen stockshave been prepared. On receipt, grow and freeze multiple vials of Expi293F™

cells to ensure that you have an adequate supply of early-passage cells.

• Allow freshly thawed cells to recover in culture for three or more passagespost-thaw before transfecting.

Required materials

• Expi293F™ cells

• 125-mL non-baffled, disposable, sterile, vent-cap shaker flask for culturingsuspension cells

• Expi293™ Expression Medium, pre-warmed to 37℃• Orbital shaker in temperature and CO2 controlled incubator

• Reagents and equipment to determine cell viability (for example, hemocytometerwith trypan blue or cell counter)

MethodsThaw and establish Expi293F™ cells


Thaw Expi293F™ cells

1. Add 30 mL of pre-warmed (37°C) Expi293™ Expression Medium into a 125-mLpolycarbonate or PETG, disposable, sterile, vented Erlenmeyer shaker flask.

2. Remove one vial of cells from liquid nitrogen, then swirl in a 37°C water bath for1 to 2 minutes to thaw the cells rapidly until only a small amount of ice remains.

IMPORTANT! Do not submerge the vial in the water.

3. Just before the cells are completely thawed, decontaminate the vial by wiping itwith 70% ethanol before opening it in a laminar flow hood.

4. Use a 2-mL or 5-mL pipette to transfer the entire contents to the flask with30 mL of pre-warmed (37°C) Expi293™ Expression Medium.

5. Incubate the cells in a 37°C incubator with ≥80% relative humidity and 8% CO2on an orbital shaker platform according to Table 2.

Note: Other CO2 settings (5%) can be used if necessary but may compromisecell health or protein production.

Table 2

Shaker diameter Shake speed (rpm)

19 mm 125 ± 5

25 mm 120 ± 5

50 mm 95 ± 5

6. Allow cells to culture for 3–4 days post-thaw, then determine viable cell densityand percent viability.

Note: Cell viability should be ≥90% 3–4 days post-thaw with viable cell densitytypically >1 × 106 viable cells/mL. Cells may be incubated for up to an additional3 days in order to reach ≥90% viability post-thaw.

7. Perform the first subculture when the viable cell density reaches 1–3 × 106 viablecells/mL (typically 4–7 days post-thaw).

8. For routine cell culture maintenance, subculture cells every 3–4 days when cellsreach 3–5 × 106 cells/mL. Do not subculture cells before reaching early logphase growth of ≥3 × 106 cells/mL.

Subculture Expi293F™ cells

Expi293F™ cells are capable of achieving high cell densities; therefore, werecommend that the cells attain a minimum density of 3–5 × 106 viable cells/mLat the time of subculturing.

MethodsSubculture Expi293F™ cells


Required materials

• Expi293F™ cell culture at 3–5 × 106 viable cells/mL

• Expi293™ Expression Medium, pre-warmed to 37°C

• Polycarborate or PETG, non-baffled, disposable, sterile, vented shaker flask forculturing suspension cells

• Reagents and equipment to determine viable cell density and percent viability(e.g., hemocytometer or an automated cell counter, trypan blue)

• Orbital shaker in a 37°C incubator with ≥80% relative humidity and 8% CO2

Passage Expi293F™ cells

1. Use the viable cell density to calculate the volume of cell suspension required toseed a new shake flask according to the recommended seeding densities in andthe recommended culture volumes in .

Table 3 Recommended seeding densities for routine cell culturemaintenance

Sub-culture timing Recommended seeding density

For cells ready 3 days post-subculture 0.4–0.6 × 106 viable cells/mL

For cells ready 4 days post-subculture 0.2–0.4 × 106 viable cells/mL

Table 4 Recommended volumes for routine cell culture maintenance invented, non-baffled flask

Flask sizeCulture

volume (mL)Shake speed

125 mL 30–35 mL

125 ± 5 rpm (19 mm shaking diameter)120 ± 5 rpm (25 mm shaking diameter)95 ± 5 rpm (50 mm shaking diameter)

250 mL 60–70 mL

500 mL 120–140 mL

1 L 240–280 mL

2 L 480–560 mL

3 L 720–840 mL90± 5 rpm (19 mm shaking diameter)85± 5 rpm (25 mm shaking diameter)80± 5 rpm (50 mm shaking diameter)

Note: If using volumes outside of the recommended ranges, it is critical toensure that all cell growth (i.e., doubling times), health (i.e., cell diameter,viability) and expression levels remain consistent with control conditions. Cellperformance will be decreased if cell health is compromised. Typically, whenusing higher volumes shaking speed will need to be increased to the upper limitof the ranges provided above.

2. Transfer the calculated volume of cells to fresh, pre-warmed Expi293™

Expression Medium in a shake flask.

MethodsSubculture Expi293F™ cells


3. Incubate flasks in a 37°C incubator with ≥80% relative humidity and 8% CO2on an orbital shaker platform until cultures reach a density of 3–5 × 106 viablecells/mL.

Note: Cells that are subcultured at densities outside of this early log-phasegrowth window may show longer doubling times and lower titers over time.Modify the initial seeding density to attain the target cell density of 3–5 × 106

viable cells/mL at the time of subculturing.

4. Repeat step 1 through step 3 to maintain or expand the cells for transfection.

Cryopreserve Expi293F™ cells

Expi293F™ cells can be frozen directly in Expi293™ Expression Medium plus DMSOor a mixture of conditioned culture medium plus DMSO. When freezing Expi293F™

cells, follow these recommendations.

1. Centrifuge cells that have attained a viable cell density of 3–5 x 106 viablecells/mL at 300 × g for 5 minutes to pellet the cells.

2. Discard the spent medium, replace the spent medium with ice-cold Expi293™

Expression Medium with 10% DMSO, then gently resuspend the cell pellet bypipetting.

3. Dilute the cells to a final density of 1 × 107 viable cells/mL in 1 mL total volumeof 90% fresh Expi293™ Expression Medium and 10% DMSO.

Note: Alternatively, conditioned medium obtained following centrifugation of thecells prior to freeze down can be added to fresh Expi293™ Expression Medium inthe following ratios: 45% fresh Expi293™ Expression Medium, 45% conditionedmedium and 10% DMSO to generate a conditioned freeze medium.

4. Freeze the cells in an automated or manual controlled-rate freezing apparatusfollowing standard procedures.

Note: For ideal cryopreservation, the freezing rate should be a decrease of 1°Cper minute.

5. Transfer frozen vials to liquid nitrogen for long-term storage.

Transfect Expi293F™ cells

For optimal transfection of high-density suspension Expi293F™ cultures, use theExpiFectamine™ 293 Reagent included in the transfection kit. Unlike some otherserum-free media formulations, Expi293™ Expression Medium does not inhibittransfection. Expi293™ Expression Medium is specifically formulated to enabletransfection without the need to change or add media.

MethodsTransfect Expi293F™ cells


Required materials

• Expi293F™ cell culture in Expi293™ Expression Medium

• Plasmid DNA, sterile, free from phenol and sodium chloride, and containingmostly supercoiled DNA

Note: We recommend isolating plasmid DNA using the PureLink™ PlasmidIsolation Kits (For ordering information, see Appendix C, “Ordering information”).To ensure sterility, DNA can be filtered through a 0.22-μm filter before use.

• Antibody-Expressing Positive Control Vector

• ExpiFectamine™ 293 Transfection Kit

• Opti-MEM™ I Reduced Serum Medium


Note: Do not add antibiotics to culture media during transfection as thiswill decrease transfection efficiency. If necessary, antibiotics can be added tocultures approximately 24 hours posttransfection.

• Disposable, sterile Erlenmeyer flasks


• Reagents and equipment to determine viable cell density and percent viability

Guidelines for transfection

• Allow freshly thawed cells to recover in culture for three or more passagespost-thaw before transfecting.

• During all cell manipulations, mix the cells by gentle swirling; avoid vigorousmixing/pipetting. Cell health is critical to maximal performance.

• Use of transfection reagents other than the ExpiFectamine™ 293 Reagent totransfect Expi293F™ cultures can lead to substantially reduced performance.

• Gently invert the ExpiFectamine™ 293 Reagent 4–5 times before use to ensurethorough mixing.

• Complexation of plasmid DNA and ExpiFectamine™ 293 Reagent takes place atroom temperature.

• Once combined, you should add the ExpiFectamine™ 293/DNA complexes to thecells after a 10–20 min incubation period. Longer hold times may lead to slightlosses in performance. Hold times over 20 minutes are not recommended.



Scale up transfections

You can scale up the Expi293F™ cultures in spinner flasks or bioreactors. Determinethe optimal spinner or impeller speed and seeding density for your culture system.We recommend that the cells be seeded at 0.3 × 106 to 0.5 × 106 viable cells/mL.Optimum spinner speed is approximately 100–130 rpm. For more information onprotein expression in 3L bioreactors, see page 18. If the split ratio of cells to freshmedia is less than 1:2, centrifuge the cell suspension and re-suspend the cell pellet infresh medium before inoculating the culture.

Use the following conditions to scale up transfections:

Table 5 Recommended volumes for transfection at various scales

Vessel type96 deep

well plate24 deep

well plate

MiniBioreactor

tube

125 mLflask

250 mLflask

1 L flask 2 L flask 3 L flask

Number of cellsrequired

2.0 × 106 7.5 × 106 45 × 106 75 × 106 150 × 106 600 × 106 1.2 × 109 2.25 × 109

Culture volume totransfect

800 µL 2.5 mL 15 mL 25 mL 50 mL 200 mL 400 mL 800 mL

Shake speed[1] (rpm)

900±50(3mmobital

shakingdiameter

225±5250±5235±5

240±5250±5245±5

125±5 (19mm orbital shaking diameter)120±5 (25mm orbital shaking diameter)95±5 (55mm orbital shaking diameter)

90±590±555±5

Amount of plasmidDNA

1.0 µg total plasmid DNA per mL of culture volume to transfect

Volume of plasmidDNA[2] 0.8 µL 2.5 µL 15 µL 25 µL 50 µL 200 µL 400 µL 800 µL

Opti-MEM™ IReduced SerumMedium[3]

50 µL 150 µL 900 µL 1.5 mL 3 mL 12 mL 24 mL 48 mL

ExpiFectamine™ 293Reagent

2.5 µL 8 µL 50 µL 80 µL 160 µL 640 µL 1.3 mL 2.6 mL

Opti-MEM™ IReduced SerumMedium[4]

50 µL 140 µL 850 µL 1.4 mL 2.8 mL 11.2 mL 22.5 mL 45 mL

ExpiFectamine™

293 TransfectionEnhancer 1

5 µL 15 µL 90 µL 150 µL 300 µL 1.2 mL 2.4 mL 4.8 mL



Table 5 Recommended volumes for transfection at various scales (continued)

Vessel type96 deep

well plate24 deep

well plate

MiniBioreactor

tube

125 mLflask

250 mLflask

1 L flask 2 L flask 3 L flask

ExpiFectamine™

293 TransfectionEnhancer 2

50 µL 150 µL 900 µL 1.5 mL 3 mL 12 mL 24 mL 48 mL

Final culture volume ~1 mL ~3 mL ~20 mL ~30 mL ~60 mL ~240 mL ~480 mL ~960 mL

[1] Recommended shake speed ranges; optimal shake speed should be determined empirically based on the specific laboratory equipment used.[2] Assuming a plasmid DNA stock concentration of 1mg/mL and a final concentration of 1.0 μg plasmid DNA per mL[3] Volume of Opti-MEM™ I Reduced Serum Medium used to dilute plasmid DNA[4] Volume of Opti-MEM™ I Reduced Serum Medium used to dilute ExpiFectamine™ 293 Reagent

Transfect Expi293F™ cells

During all cell manipulations, mix the cells by gentle swirling; avoid vigorousmixing/pipetting. Cell health is critical to maximal performance.

Refer to Table 3 for suggested volumes for transfection at various scales.

1. Subculture and expand Expi293F™ cells until the cells reach a density ofapproximately 3–5 × 106 viable cells/mL.

Day −1: Split cells2. On the day prior to transfection (Day −1), split the Expi293F™ culture from Step

1 to a final density of 2.5 –3 × 106 viable cells/mL and allow the cells to growovernight.

Day 0: Transfect cells3. On the next day (Day 0), determine viable cell density and percent viability.

The cells should have reached a density of approximately 4.5–5.5 × 106 viablecells/mL. Viability should be 95–99% to proceed with transfection.

4. Dilute the cells from Step 2 to a final density of 3 × 106 viable cells/mL with freshExpi293™ Expression Medium, pre-warmed to 37°C. Swirl the flasks gently tomix the cells.

Note: Discard the remaining cells; do not re-use high-density cells for routinesubculturing.

5. Prepare ExpiFectamine™ 293/plasmid DNA complexes as described (see Table 5for recommended volumes).

Note: Total plasmid DNA of 1.0 µg per mL of culture volume to be transfected isappropriate for most proteins.

a. Gently invert the ExpiFectamine™ 293 Reagent bottle 4–5 times to mix.

b. Dilute plasmid DNA with Opti-MEM™ I Reduced Serum Medium. Mix byswirling the tube and/or by inversion.



c. Dilute ExpiFectamine™ 293 Reagent with Opti-MEM™ I Reduced SerumMedium. Mix by swirling the tube and/or by inversion or gentle pipetting2–3 times and allow to incubate at room temperature for 5 minutes prior toinitiating the plasmid DNA complexation reaction.

d. Add the diluted ExpiFectamine™ 293 Reagent to diluted plasmid DNA. Mixby swirling the tube and/or by inversion or gentle pipetting 2–3 times.

6. Incubate ExpiFectamine™ 293/plasmid DNA complexes (from Step 5d) at roomtemperature for 10–20 minutes, and then slowly transfer the solution to theshaker flask from Step 4, swirling the flask gently during addition.

7. Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO2in air on an orbital shaker (for suggested shake speeds, see Table 4).

Day 1: Add ExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293 TransfectionEnhancer 2

8. On the day after transfection (Day 1, 18–22 hours post-transfection), addExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293Transfection Enhancer 2 to the flask (see Table 5), gently swirling the flask duringaddition. Return the flask to the 37°C incubator with a humidified atmosphere of8% CO2 with shaking.

Note: It is not necessary to pre-warm ExpiFectamine™ 293 TransfectionEnhancer 1 and ExpiFectamine™ 293 Transfection Enhancer 2 prior to addition toflasks.

Note: ExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293Transfection Enhancer 2 may be pre-mixed together just prior to adding to flasksfor convenience.

Day 5:9. Optimal time to harvest protein will depend on the specific properties of the

protein being expressed. 5–7 days post-transfection is a typical harvest time toreach maximum titers for many secreted proteins. For membrane proteins, 3–4days is a typical harvest time.



Protein expression in 3L Bioreactors

Required materials

• Expi293F™ cell culture in Expi293™ Expression Medium

• Plasmid DNA preparation, sterile, free from phenol and sodium chloride, andcontaining mostly supercoiled DNA

Note: We recommend isolating plasmid DNA using the PureLink™ kits (Forordering information, see Appendix C, “Ordering information”). To ensure sterility,DNA can be filtered through a 0.22-μm filter before use.

• Opti-MEM™ I Reduced Serum Medium

Note: If transfection under AOF (animal origin free) conditions is desired, Opti-MEM™ I can be replaced with Opti-Plex™ Complexation Buffer.


Note: Do not add antibiotics to culture media during transfection as thiswill decrease transfection efficiency. If necessary, antibiotics can be added tocultures approximately 24 hours post-transfection.

• Polycarborate or PETG, non-baffled, disposable, sterile, vented shaker flask forculturing suspension cells


• 3L HyPerforma™ Glass Bioreactor with HyPerforma™ G3Lab Controller orcomparable

• Nalgene™ PETG bottles with transfer caps or other transfer bottles

• Sterile Tube Welder

• Reagents and equipment to determine viable cell density and percent viability

• Reagents and equipment to determine gas concentrations, pH, and metabolites

Recommended volumes for 3L Bioreactor transfection

Table 6

Reagent Volume

Culture volume to transfect 1.8 L

Amount of plasmid DNA required 1.0 mg total plasmid DNAper liter of culture to

transfect

Volume of plasmid DNA[1] required 1.8 mL

Volume of Opti-MEM™ I Reduced Serum Medium or Opti-Plex™ Complexation Buffer required to dilute plasmid DNA

90 mL

Volume of ExpiFectamine™ 293 Reagent 5.8 mL

MethodsProtein expression in 3L Bioreactors


Table 6 (continued)

Reagent Volume

Volume of Opti-MEM™ I Reduced Serum Mediumor Opti-Plex™ Complexation Buffer required to diluteExpiFectamine™ 293 Reagent

90 mL

ExpiFectamine™ 293 Transfection Enhancer 1 Volume 10.8 mL

ExpiFectamine™ 293 Transfection Enhancer 2 Volume 108 mL

Final culture volume ~2 L

[1] Assuming a plasmid DNA stock concentration of 1mg/mL.

Recommended Bioreactor settings

Table 7

Parameter Setting

Temperature 37℃ ± 0.5

Working Volume 2 L

Sparger L – Shaped Drilled Hole macrosparge

Impellers 1 x Rushton (bottom), 1 x three pitched blade (top)

Impeller Diameter 55 mm

Impeller Power Number 1.4

Agitation 140 RPM, P/V 4.5 W/m3, tip speed 0.4 m/s

Headspace Gassing Air - 0.05 lpm

Dissolved Oxygen (DO) 40%

pH for Growth ≤7.25 controlled by CO2

pH for Production 6.80 ± 0.05

Transfect Expi293F™ Cells in a 3L Bioreactor

1. Subculture and expand Expi293F™ cells until they reach a density of 3–5 × 106

viable cells/mL in a culture volume that will yield at least 9 × 108 viable cells toinoculate one bioreactor.

Note: Allow cells to recover for at least three passages after thaw beforetransfecting.

2. Calibrate DO and pH probes and add to assembled bioreactor.

3. Sterilize bioreactor and allow to cool to room temperature.



4. Add 600 mL Expi293™ Expression Medium to the bioreactor. Turn on agitation,headspace gassing, and temperature control. Allow medium to equilibrate totemperature set point before seeding cells.

5. Seed bioreactor by adding Expi293F™ cells from step 1 to a final cell density of 1× 106 viable cells/mL in a total volume of 900 mL.

6. Check calibration of pH and perform a 1 point calibration offset if needed.

7. Three days later, on the day of transfection, determine viable cell density andviability. Cells should have reached approximately 3.5–5.5 × 106 viable cells/mL.Viability should be >95% to proceed with transfection.

8. Add pre-warmed Expi293™ Expression Medium to the bioreactor to dilute thecells to a final density of 3 × 106 viable cells/mL.

9. Adjust pH set point to 6.8 ± 0.05 controlled by CO2. Allow bioreactor toequilibrate.

10. Prepare ExpiFectamine™ 293 Reagent/plasmid DNA complexes.

a. To a 250 mL Nalgene™ PETG bottle, add Opti-MEM™ I Reduced SerumMedium or Opti-Plex™ Complexation Buffer at 5% of the volume of cellculture to be transfected (for example, 90 mL for transfecting 1.8 L ofculture).

b. Gently invert the ExpiFectamine™ 293 Reagent bottle 4–5 times to mix.

c. Add ExpiFectamine™ 293 Reagent to a final concentration of 3.2 mL/L ofculture to be transfected to the bottle in substep 10a (for example, add5.8 mL ExpiFectamine™ 293 Reagent to transfect 1.8 L of culture).

d. To a second 250 mL Nalgene™ PETG bottle, add Opti-MEM™ I ReducedSerum Medium or Opti-Plex™ Complexation Buffer at 5% of the volume ofcell culture to be transfected (for example, 90 mL for transfecting 1.8 L ofculture).

e. Add plasmid DNA to a final concentration of 1.0 mg/L of culture to betransfected to the second 250mL Nalgene™ PETG bottle (for example, add1.8 mg of DNA to transfect 1.8 L of culture).

f. Combine the two bottles by adding the diluted transfection reagent fromsubstep 10c to the diluted DNA.

11. Incubate the ExpiFectamine™ 293 Reagent/plasmid DNA complex at roomtemperature for 10 minutes.

Following the incubation attach a transfer cap to the bottle, sterile tube weld thebottle to the bioreactor, then use a peristaltic pump to transfer the solution intothe bioreactor.



Note: For optimal performance, the ExpiFectamine™ 293 Reagent/plasmid DNAcomplex should be fully added to the bioreactor within 20 minutes of combiningthe contents of the two bottles.

12. Eighteen to 22 hours post-transfection:

a. Add ExpiFectamine™ 293 Transfection Enhancer 1 at a final volume of0.6% v/v (6 mL/L) of culture volume transfected (for example, add 10.8mL ExpiFectamine™ 293 Transfection Enhancer 1 per 1.8 L of culturetransfected)

b. Add ExpiFectamine™ 293 Transfection Enhancer 2 to a final volumeof 6% (60 mL/L) of culture volume transfected (for example, 108mL ExpiFectamine™ 293 Transfection Enhancer 2 per 1.8 L of culturetransfected).

13. Sample bioreactor daily.

• Count cells on a cell counter.

• Check pH, O2, and CO2 on a gas analyzer.

• Check metabolites on a bioanalyzer.

• Aseptically remove samples for analysis to determine optimal harvest time,approximately 48 hours post transfection.

Additional guidelines

• For optimal performance, it is critical that the cell density, shaking diameter,shaking speed, flask size/type and volume of culture to be transfected match therecommendations in Table 3 and Table 4 above for routine maintenance of cells.

• Ensure all equipment is calibrated prior to use. Out of specification temperature,gas volumes, and pH control can negatively affect the system and lead toreduced titers, decreased cell growth, clumping or cell death.

Guidelines to scale up into larger vessels

• For scale up into larger vessels above a 2 L working volume considerationshould be taken for power input per volume (P/V), tip speed, mixing time andaddition of the transfection complex. The P/V for the listed protocol is 4.5 W/m3

and the tip speed is 0.4 m/s. This provides a robust system at the 2 L workingvolume scale that minimizes shear from the impeller while allowing for optimalmixing. We recommend to scale up using P/V when possible.

• When larger scales are being considered attention should be given to hold timeand mixing of transfection reagents, DNA, and the transfection complex. Planahead to ensure each step is thoroughly mixed and incubation/addition timing isnot prolonged unnecessarily.

• It should also be noted that a decrease in titer is not uncommon withincreased scale. Minimizing hold and addition times, ensuring proper mixing ofthe transfection complex, and optimization of process parameters should helpminimize this decrease.



Additional guidelines

Guidelines to optimize protein expression

• Expression levels will vary depending on the specific recombinant proteinexpressed and the vector used; however, the Expi293™ Expression Systemwill exhibit consistent expression level for any particular protein from onetransfection to the next.

• When expressing a protein for the first time, you may want to perform a timecourse (e.g., harvest cells or media at several time points posttransfection) tooptimize the length of the expression run.

• When expressing antibody molecules with the heavy and light chains encodedon two separate plasmids, we also recommend optimizing the ratio of heavychain to light chain for each individual antibody. We recommend initial testing ofheavy chain:light chain ratio at 1:2.

Equipment

• For optimal performance, it is critical that the shaking diameter, shakingspeed, flask size/type, and volume of culture to be transfected match therecommendations in this protocol for both routine subculture and proteinexpression runs.

• Humidified incubators (≥80% relative humidity) are recommended to reduceevaporation during expression runs. When using multi-well plates, high-humiditysettings should be used if available, as evaporation will be greater.

• Ensure equipment is calibrated for temperature. In some instances, the total heatfrom the incubator and the shaker can cause cell culture temperatures to exceedthe recommended ranges and lead to decreased cell growth, clumping or celldeath. In such instances, reduce the temperature setting of the incubator tocompensate for heat generated by the shaker.

• Ensure that equipment is calibrated for CO2. Levels of CO2 should not exceed8%.

A


Cells

• Cells should recover rapidly post-thaw and exhibit growth profiles within theguidelines of the protocol during routine cell culture maintenance for 3–4 days(see “Expi293F™ Cells” on page 6).

• Expi293F™ is a high-density cell line: subculture cells when density has reachedlog phase growth at 3–5 × 106 viable cells/mL. Subculturing cells before theyhave reached log phase growth can negatively impact cell performance.

• During all cell manipulations, mix the cells by gentle swirling; avoid vigorousmixing/pipetting, especially immediately before transfection. Cell health prior totransfection is critical to maximal performance.

• Always keep dedicated cell culture maintenance flasks: do not re-purposeremaining high-density cells from a transfection run for routine subculturing.

Plasmid DNA complexation

• Plasmid DNA is highly stable in Opti-MEM™ I Reduced Serum Medium. OnceExpiFectamine™ 293 Reagent is diluted with Opti-MEM™ I Reduced SerumMedium mix by swirling the tube and/or inversion or gentle pipetting 2–3 times.Do not vortex.

• For optimal performance, once the diluted ExpiFectamine™ 293 Reagent isadded to diluted plasmid DNA, mix by swirling the tube and/or inversionor gentle pipetting 2–3 times; do not vortex. Incubate 10–20 minutes post-complexation before drop-wise addition to the flasks with swirling.

Harvest

• For typical proteins, high cell viability (ideally 65–75% or greater) is observed atthe time of protein harvest (i.e., 5–7 days).

Cell culture supernatant clarification

• Following harvest, centrifuge the supernatant at 3000–5000 x g for 20–30minutes in a refrigerated centrifuge.

• Filter supernatant through a 0.22-μm filter.

Appendix A Additional guidelinesCells A


Positive control for transfection andexpression

Antibody-Expressing Positive Control Vector

Antibody-Expressing Positive Control Vector is provided as a positive control fortransfection and expression in Expi293™ cells. The control contains pcDNA3.4plasmid clones expressing the heavy and light chains of a rabbit IgG. The control isprovided as a ready-to-use transfection-grade plasmid at a concentration of 1 mg/mLwith a 1:2 heavy chain: light chain ratio and is sufficient to transfect up to 150 mL ofExpi293™ cells.

Transfection and expression

Transfect 25 mL of suspension Expi293™ cells using 25 µL of the Antibody-Expressing Positive Control Vector (i.e., 1 µg of positive control per 1 mL of Expi293™

culture) following the protocol provided in the Transfecting Expi293F™ cells section.

The rabbit IgG that is produced in Expi293™ cells after transfection with the controlvector is secreted into the Expi293™ Expression Medium, with optimal yieldsobtained between 5–7 days.

Note: The titer values referenced above were determined in crude cell culturesupernatants using a Pall Life Sciences FortéBio™ Octet™ instrument equipped witha protein A biosensor.

B


Ordering information

Unless otherwise indicated, all materials are available through thermofisher.com.

Additional products

Item Amount Source

Expi293F™ cells1 mL

A14527A14527CN

6 × 1 mL A14528

Expi293F™ Cells (cGMP banked) 1 vial 100044202

ExpiFectamine™ 293 Transfection Kit1 kit for 1 L of

cultureA14524

Expi293™ Expression Medium 1 L A1435101

Opti-Plex™ Complexation Buffer 100 mL A4096801

Expi293F™ Inducible Cells 1 mL A39241

Expi293F™ GnTI- Cells 1 mL A39240

Expi293F™ Inducible GnTI- Cells 1 mL A39242

Expi293™ Met (-) Protein Labeling Kit 1 kit A41249

ExpiFectamine™ 293 Met (-) TransfectionKit

1 kit for 1 L ofculture

A39249

Expi293™ Met (-) Expression Medium 1 L A4096701

pRABBIT IgG IRES-EmGFP PositiveControl Vector

1 kit A39243

Antibody-Expressing Positive ControlVector

1 vial A14662

PNGase F Glycan Cleavage Kit 1 kit (500,000 units) A39245

pcDNA™5/TO Mammalian ExpressionVector

1 kit V103320

pcDNA™ 3.4-TOPO™ TA Cloning Kit 1 kit A14697

L-Methionine (Methyl-13C) 225 mg A39248

C


http://www.thermofisher.com

(continued)

Item Amount Source

L-Selenomethionine 250 mg A39247

Tetracycline Hydrochloride 500 mg A39246

Trypan Blue Stain 100 mL 15250-061

Shaker flasks for suspension culture

Item Capacity Source

Nalgene™ Single-Use PETG ErlenmeyerFlasks with Plain Bottom: Sterile

125 mL 4115-0125

250 mL 4115-0250

500 mL 4115-0500

1,000 mL 4115-1000

2,000 mL 4115-2000

2,800 mL 4115-2800

Orbital shaker

Item Source

MaxQ™ HP Tabletop Orbital Shaker SHKE416HP

CO2 controlled incubator

Item Source

Large-Capacity Reach-In CO2 Incubator 3950

Appendix C Ordering informationShaker flasks for suspension cultureC


Plasmid purification products

Item Amount Source

PureLink™ HiPure Plasmid Midiprep Kit 25 preps K210004

PureLink™ HiPure Plasmid Filter Midiprep Kit 25 preps K2100-14

PureLink™ HiPure Plasmid Maxiprep Kit 10 preps K210006

PureLink™ HiPure Plasmid Filter Maxiprep Kit 10 preps K2100-16

PureLink™ HiPure Expi Plasmid Megaprep Kit 4 preps K210008XP

Visualization and quantitation or control antibody

Item Amount Source

Protein A 25 mg 101006

F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-AdsorbedSecondary Antibody, HRP

500 µg A10547

Rabbit IgG Isotype Control 10 mg 02-6102

SimplyBlue™ SafeStain 1 L LC6060

NuPAGE™ 4–12% Bis-Tris Protein Gel, 1.0 mm, 12-well (10 gels/box)

1 box NP0322BOX

Appendix C Ordering informationPlasmid purification products C


Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specified inthe user documentation may result in personal injury or damage to the instrumentor device. Ensure that anyone using this product has received instructions ingeneral safety practices for laboratories and the safety information provided in thisdocument.

· Before using an instrument or device, read and understand the safety informationprovided in the user documentation provided by the manufacturer of theinstrument or device.

· Before handling chemicals, read and understand all applicable Safety Data Sheets(SDSs) and use appropriate personal protective equipment (gloves, gowns, eyeprotection, and so on). To obtain SDSs, see the “Documentation and Support”section in this document.

D


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensurelaboratory personnel read and practice the general safety guidelines for chemicalusage, storage, and waste provided below. Consult the relevant SDS for specificprecautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by the chemicalmanufacturer before you store, handle, or work with any chemicals or hazardousmaterials. To obtain SDSs, see the "Documentation and Support" section in thisdocument.

· Minimize contact with chemicals. Wear appropriate personal protective equipmentwhen handling chemicals (for example, safety glasses, gloves, or protectiveclothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open. Useonly with sufficient ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, follow themanufacturer cleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.

· Ensure use of primary and secondary waste containers. (A primary waste containerholds the immediate waste. A secondary container contains spills or leaks fromthe primary container. Both containers must be compatible with the waste materialand meet federal, state, and local requirements for container storage.)

· After emptying a waste container, seal it with the cap provided.

· Characterize (by analysis if needed) the waste generated by the particularapplications, reagents, and substrates used in your laboratory.

· Ensure that the waste is stored, transferred, transported, and disposed ofaccording to all local, state/provincial, and/or national regulations.

· IMPORTANT! Radioactive or biohazardous materials may require special handling,and disposal limitations may apply.

WARNING! HAZARDOUS WASTE (from instruments). Waste produced by theinstrument is potentially hazardous. Follow the guidelines noted in the precedingGeneral Chemical Handling warning.

WARNING! 4L Reagent and Waste Bottle Safety. Four-liter reagent and wastebottles can crack and leak. Each 4-liter bottle should be secured in a low-densitypolyethylene safety container with the cover fastened and the handles locked in theupright position.

Appendix D SafetyChemical safety D


Biological hazard safety

WARNING! Potential Biohazard. Depending on the samples used on thisinstrument, the surface may be considered a biohazard. Use appropriatedecontamination methods when working with biohazards.

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. Conduct all work in properly equipped facilities withthe appropriate safety equipment (for example, physical containment devices). Safetyequipment can also include items for personal protection, such as gloves, coats,gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles.Individuals should be trained according to applicable regulatory and company/institution requirements before working with potentially biohazardous materials.Follow all applicable local, state/provincial, and/or national regulations. The followingreferences provide general guidelines when handling biological samples in laboratoryenvironment.

· U.S. Department of Health and Human Services, Biosafety in Microbiological andBiomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112,Revised December 2009; found at:https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf

· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Appendix D SafetyBiological hazard safetyD


https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf

https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Documentation and support

Customer and technical support

Visit thermofisher.com/support for the latest service and support information.

• Worldwide contact telephone numbers

• Product support information– Product FAQs

– Software, patches, and updates

– Training for many applications and instruments

• Order and web support

• Product documentation– User guides, manuals, and protocols

– Certificates of Analysis

– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products asset forth in the Life Technologies' General Terms and Conditions of Saleat www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If youhave any questions, please contact Life Technologies at www.thermofisher.com/support.

E


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